<p>Sight-threatening diseases, such as age-related macular degeneration (AMD), affect the tissues of the posterior segment of the eye. Though modern classes of biomolecular based drugs are therapeutically useful, drug targeting for prolonged bioavailability to pathological sites within the eye is challenging. Current delivery approaches are invasive and lack control over drug release rates and tissue-specific localization. In this thesis, a device using microneedles embedded in a flexible platform was developed that could potentially overcome these challenges.</p> <p>New methods for microneedle fabrication were developed by co-opting simple chemical etch methods commonly used for optical probe fabrication as an alternative to current complex and expensive photolithographic technologies to produce out-of-plane, high aspect ratio microneedles which are often constrained materially to silicon and metal. Microneedles with repeatable tip and taper sizes were obtained using hydrofluoric acid, an organic phase and fused-silica capillary tubing. Microneedles with 10 um tips were made using single and batch mode methods and were then integrated into poly (dimethylsiloxane) (PDMS) for alignment using low cost micromolding approaches offering the same degree of accuracy provided by conventional photolithography<strong>. </strong></p> <p>Single microneedle-based devices successfully delivered rhodamine intrasclerally, intravitreally, suprachoroidally and to the retina. This is the first demonstration of active delivery to specific spatial regions within the posterior eye at controllable rates using a non-implantable, biocompatible device – with minimal fabrication facilities, equipment and cost. The fabricated device demonstrated a new hybrid approach of coupling a rigid microneedle with a soft and pliable substrate that could conform to biological tissues.</p> / Doctor of Philosophy (PhD)

<p>The mechanical and chemical properties of hydrogels make them excellent vehicles to deliver drugs. However, current systems encounter difficulties with loading hydrophobic molecules into the aqueous gel network and the subsequent release of the drug from the gel matrix. Cyclodextrins (CDs) offer a potential solution to this drug delivery challenge. CDs have the unique property of possessing a hydrophilic exterior and a hydrophobic interior pocket which is capable of hydrophobic drug binding. CD molecules complexed with hydrophobic drugs have been demonstrated to significantly increase the bioavailability of those drugs in free solution. Thus, if these nanodomains are introduced into microgels or hydrogels, we anticipate that significantly higher hydrophobic drug loadings may be achieved together with improved controlled release of these drugs based on the properties of the hydrogel or microgel phase. We have fabricated <em>in situ</em> gellable and degradable hydrogels and microgels based on combinations of CDs and either functionalized carbohydrates (dextran) or thermosensitive synthetic polymers (poly(N-isopropylacrylamide), PNIPAM). To achieve this goal, we designed a series of microgels with grafted or immobilized CD groups and used multi-functional CD as a reactive crosslinker for making injectable bulk hydrogels.</p> / Master of Applied Science (MASc)

<p>Collagen crosslinking with a polypropyleneimine octaamine dendrimers, via carbodiimide chemistry, was further exploited to demonstrate the ability of this technology for various tissue engineering strategies, including tissue engineered corneal equivalents (TECE) and blood-contacting biomaterials. In addition, modification with extracellular matrix components and other biomimetic molecules may enhance tissue-host interactions for greater <em>in vivo </em>compatibility.</p> <p>First, the efficacy of the dendrimer crosslinking technology was further validated with commercially available collagen-based materials, from bovine or human sources (Chapter 4: Paper 1), as determined via transmittance, water uptake, differential scanning calorimetry, collagenase stability and <em>in vitro </em>cell compatibility. Despite gel formation, the matrix integrity was compromised with collagen-based materials manufactured under acidic conditions and purified via freeze-drying.</p> <p>To continue the theme of dendrimer crosslinked collagen gels as TECE materials, growth factor incorporation was investigated with epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), as a method for improving device epithelialization and subsequent host integration. However, given the short half lives of these growth factors, an effective growth factor delivery system is necessary to protect growth factor bioactivity. As heparan sulphate proteoglycans sequester and release heparin-binding growth factors <em>in vivo</em>, the use of heparinized dendrimer crosslinked collagen (CHG) gels for HB-EGF delivery would provide prolonged, controlled delivery, while maintaining growth factor effectiveness (Chapter 5: Paper 2). HB-EGF release was prolonged and capable of inducing human cornea epithelial cell (HCEC) proliferation. Thus, HB-EGF delivery from CHG gels could aid in TECE device retention through enhanced device-host integration via epithelialization.</p> <p>Alternatively, tethering EGF or HB-EGF to dendrimer crosslinked collagen (CG) gels could also supply growth factor stimulation in a manner that maintains bioactivity, while stimulating growth factor receptors continually with minute concentrations (Chapter 6: Paper 3). Growth factor uptake and bioactivity was assessed with radiolabeled growth factor and through <em>in vitro </em>epithelial cell culture, respectively. Surface-modification of CG gels with growth factors demonstrated greater bioactivity, compared to growth factor bulk-modification of CG gels.</p> <p>Finally, dendrimer crosslinked collagen gels, with pre-activated heparin (PH gels) were investigated as a tissue engineered blood-contacting biomaterial (Chapter 7: Paper 4), as we hypothesized that biomaterial induced coagulation is not only influenced by an anticoagulant surface, but also by the underlying material and that improved blood-biomaterial interactions may be achieved by utilizing a natural polymer that emulates biomimetic properties. Pre-activation of heparin was utilized to increase heparin gel content, while antithrombotic properties were evaluated via antithrombin and fibrinogen adsorption and plasma recalcification times. PH gels had increased heparinization, but extensive crosslinking compromised antithrombin-heparin interactions, compared to CHG gels. CHG gels demonstrated improved antithrombotic properties and further evaluation of these gels for blood-contacting applications is warranted.</p> / Doctor of Philosophy (PhD)

<p>Coated calcium-alginate beads are the basis of many encapsulation methods used in pursuit of cell-based enzyme and hormone replacement therapies. The standard alginate - poly-L-lysine - alginate (APA) capsules consist of a calcium-alginate hydrogel core containing cells designed to express a therapeutic product, coated with permeability controlling poly-L-lysine (PLL, a polycation) followed by an exterior layer of polyanionic alginate. Although this approach is promising, the required long-term survival of the implanted cells has remained largely elusive as the current APA capsules suffer from several biocompatibility and mechanical strength issues, one of which is the weakening of ionic crosslinks over time, exposing the encapsulated cells to the host.</p> <p>This thesis aims to replace the exterior layer of alginate with a Temporarily Reactive Polyelectrolyte (TPR) to reinforce AP capsules by forming covalently crosslinked shells. TRPs are polyanions that possess reactive electrophilic groups capable of forming permanent covalent crosslinks with the underlying polyamine (such as PLL), and subsequently hydrolyze, increasing the net negative charge of the polyanion. TRPs are thought to improve the biocompatibility and strength of the microcapsules by forming stable inert amide bonds, as well as increasing the net negative charge of the capsule through the liberation of carboxylates. This thesis will focus primarily on two TRPs: 50% hydrolyzed poly(methyl vinyl ether-<em>alt</em>-maleic anhydride), PMM₅₀ , and poly(methacrylic acid-co-2-vinyl-4,4-dimethylazlactone) with a 50:50 co-monomer ratio, PMV₅₀ . Their synthesis, rates of hydrolysis and capsule formation around encapsulated C2C12 cells for <em>in-vitro</em> and<em> in-vivo</em> studies will be described. Additionally the synthesis and rates of hydrolysis of other 2-vinyl-4,4-dimethylazlactone (VDMA)-copolymers are presented as potential candidates for future TRPs.</p> / Doctor of Philosophy (PhD)

<p>Bacterial biofilms are problematic in a variety of industries hence strategies for their mitigation have received significant attention. The approach described herein attempts to control bacterial adhesion using silicone-based polymers- (widely used due to their interesting properties)- via manipulation of their surface chemistry to eventually create anti-fouling surfaces. This involved study of the systematic variation of surface wettability and its effect on <em>Escherichia coli</em> (<em>E. coli</em>) adhesion to novel polymers of acrylate-modified silicone surfactant (ACR) with either hydroxyethyl methacrylate (a hydrophilic monomer), or methyl and butyl methacrylate (hydrophobic monomers). It was hypothesized that the systematic variation of ACR would produce surfaces with differing wettability, without changing other surface properties that influence cellular adhesion. Average light transmittance across the range of visible light wavelengths (400-740nm), surface roughness and Shore 00 hardness data were consistent across the ACR-HEMA copolymer series (80-90%, ~2.5 – 5 nm, and 75-95 Shore durometer points, respectively). The same consistency was observed for surface wettability (contact angles = 78-92°) despite varying HEMA content and consequently <em>Escherichia coli</em> (<em>E.coli</em>) adhesion, likely due to system saturation with silicon (as confirmed by EDX). However, wettability of the ACR-MMA-BMA polymers did vary; ≤ 20 wt% and ≥ 80 wt% ACR polymers had contact angles between 67°- 77°, while 20 < x < 80 wt% ACR polymers had increased surface wettability (contact angles 27.6°- 42.9°). <em>E. coli </em>adhesion across the set increased with increasing ACR content, a trend mirrored by the water uptake of the materials but not the contact angle data. These results indicate that <em>E. coli </em>adhesion occurs independently of wettability for these materials and although the effect of the latter on adhesion cannot be deduced, the possible correlation between bacterial adhesion and water uptake suggests that the best antifouling surfaces should not be of materials capable of imbibing significant amounts of water.</p> / Master of Applied Science (MASc)

<p>The work presented in this thesis was focused on design and construction of novel cell-loaded microcapsules by incorporation of bioactive molecules (proteins or peptides) for potential application in hemophilia B treatment. The objective of this study was to improve the viability and functionality of the encapsulated cells by creating biomimetic microenvironments for cells that more closely mimic their physiological extracellular matrix (ECM) environment.</p> <p>Three cell-adhesive molecules were used in this work: fibrinogen and fibronectin, two abundant proteins present in ECM, and arginine-glycine-aspartic acid (RGD) tri-peptide, the minimal essential cell adhesion peptide sequence and the most widely studied peptide for cell adhesion. Alginate, the most commonly used biomaterial used for cell encapsulation, was combined with either of these molecules to create biomimetic microcapsules. Non-modified alginate (control) and modified alginate matrices were used to encapsulate the factor IX (FIX) secreting cells for protein delivery. In this work, FIX-engineered cord blood-derived human mesenchymal stem cells CB MSCs were used as a cell source for FIX delivery.</p> <p>Our data suggested that fibrinogen-alginate, fibronectin-alginate and RGD-alginate microcapsules improved the viability of encapsulated MSC and are applicable in cell therapy technologies. However, fibrinogen-alginate and fibronectin-alginate microcapsules more significantly enhanced the proliferation and protein secretion from the encapsulated cells and may have potential for FIX delivery for hemophilia B and other inherited or acquired protein deficiencies. RGD-alginate microcapsules can v potentially be used for other tissue engineering applications with the aim of enhanced viability and attachment of the enclosed cells. Differentiation studies showed the osteogenic (but not chondrogenic or adipogenic) differentiation capability of FIX-engineered CB MSCs and their efficient FIX secretion while encapsulated in fibrinogen-alginate and fibronectin-alginate microcapsules.</p> / Doctor of Philosophy (PhD)

<p>Despite the long history of topical eye drops and their use in delivering therapeutic agents to the anterior of the eye, efficient sustained delivery continues to be an elusive goal. The robust and effective clearance mechanisms that the eye is endowed with are significant delivery challenges and result in short drug residence times and low ocular bioavailability. The work carried out in this thesis focused on developing, synthesizing and characterizing silicone hydrogels and evaluating their potential as drug eluting inserts for more effective delivery of ocular pharmaceuticals. The first strategy (Chapter 2) focused on incorporating a novel hydrogel additive, hyaluronic acid, to promote hydrogel-drug ionic interactions that can function to increase drug loading and subsequent release dosage. Hydrogels composed of a hydrophilic monomer, N,N-dimethlacrylamide (DMA) or 2-hydroxyethyl methacrylate (HEMA), and a hydrophobic monomer, methacryloxypropyltris(trimethylsiloxy)silane (TRIS), were used as model contact lenses. By combining ionic interactions with molecular imprinting techniques within a single hydrogel, it was shown that this can produce a compound effect on drug uptake and release. Although greater control over release dosage was achieved, there was limited capacity for these materials to delivery timolol for extended periods with drug release occurring rapidly over a period of 1-2 days. However, there were clear differences in the release duration from the p(DMA-<em>co</em>-TRIS) and p(HEMA-<em>co</em>-TRIS) hydrogel formulations. Therefore, the second study (Chapter 3) aimed to better understand the relationship between the hydrogel chemical composition and the resultant material properties on the drug release characteristics. A range of hydrogels were synthesized with varying hydrophilic and hydrophobic monomers, which were then characterized by their water content, transparency, optical haze and surface wettability. The previous generation materials were evolved by incorporating a modified siloxy methacrylate TRIS(OH), a methacrylated polydimethylsiloxane macromonomer (mPDMS) and a polymerizable silicone surfactant (ACR). The properties of the hydrogels were dramatically affected by the nature and relative contribution of hydrophobic and hydrophilic monomers. The release of dexamethasone (DEX), an anti-inflammatory medication, was shown to vary significantly depending on the hydrogel formulations; often displaying faster release in high water content materials and slow release in low water content hydrogels. The mechanism of diffusion for lipophilic DEX in these hydrogel systems appeared to be through the internal aqueous network channels within the bulk. Over the range of hydrogels formulations that were tested, the release from them varied from approximately seven days to greater than two weeks.</p> / Master of Applied Science (MASc)

Damage to dura mater may occur during intracranial or spinal surgeries, which can result in cerebrospinal fluid leakage as well as other potentially fatal physiological changes. As a result, biological scaffolds derived from xenogeneic materials are typically used to repair and regenerate dura mater post intracranial or spinal surgeries. In this study we explore the mechanics, structure, and immunological capacity of xenogeneic dura mater to be considered as a replacement for human dura. A comparative analysis is done between native porcine dura and a commercially available bovine collagen-based dura graft. Native porcine dura mater was decellularized and subjected to mechanical and histological analysis. Our decellularized porcine dura was able to maintain the overall morphological/structural integrity and held an increased extensibility without sacrificing strength, which provides a solid foundation as a functional grafting material. The histological observations showed that the orientation of fibers was maintained after decellularization. We investigated the biocompatibility of native and decellularized porcine dura reseeded with fibroblast cells for in vitro study. Cell proliferation, cell viability, and mechanical properties of dural grafts were evaluated post reseeding on days 3, 7, and 14. Live-dead staining and resazurin salts quantified cell viability and cell proliferation, respectively. This in vitro study showed that the acellular porcine dural graft provided a favorable environment for rat fibroblast cell infiltration. The results of micro indentation testing show that the cell-seeded porcine dural graft provides a favorable environment for rat fibroblast cell infiltration. The mechanics and biocompatibility results provide promising insight for the potential use of porcine dura in future cranial dura mater graft applications. Lastly, a subcutaneous in vivo study of dura graft compared with the market available Lyoplant®. Grafts were evaluated for inflammation by evaluating macrophage and leukocyte invasion on 3, 7, and 14 days post implantation. Histological analysis of both implants revealed macrophage (and leukocyte infiltration, supporting reabsorption, and thus encouraging the regeneration at 14 days. Cell markers also revealed that inflammation and leukocytes decreased as the number of days increased. Future work will involve a long-term subcutaneous implantation up to 30 days and 60 days to determine the long-term immune response.

The body’s humoral immune response plays a larger role in the body’s defenses beyond screening for invading pathogens. Modulation of this response is also vital for tissue regeneration, drug delivery, and vaccine development. The immune system operates within a complicated feedback loop and as such, altering the strength of the immune response can be approached from an engineering perspective. While a strong initial input can direct the response to either a pro- or anti-inflammatory bias, extreme responses can be deleterious, as in the case of allergic reactions or sepsis. Therefore, the objective of this thesis was to develop a novel biomaterials platform that can be used to alter the immune response in a tunable manner. Antibodies are not only the workhorses of the adaptive immune response but are also powerful immunomodulators through their Fc (constant fragment) regions. By coating microparticles with Fc ligands in variable surface densities, we were able to utilize the sensitivity of multivalent signaling to tune the response of the immune response. Microparticle size was also varied to decouple the effects of physical versus biochemical signaling. The goal of this thesis was to analyze the effects of Fc coated particles on two major components of the humoral immune responses: macrophages and the complement system. We first looked at the mechanical response of macrophages through phagocytosis and found that both Fc density and microparticle size had significant impacts on macrophage phagocytosis. These results also provide a particle delivery “toolbox” for future applications. We then analyzed the downstream effects of Fc particles on macrophage phenotype and on phenotype plasticity. This showed that the addition of Fc particles lead to increased production of TNFα and IL-12 and inverted the response of LPS treated macrophages. Finally, we applied our particles to activate the complement system, an often overlooked cascade of serum protein activation that results in bacterial cell lysis. Cleaved components of the complement system are also powerful chemokines and can act as a vaccine adjuvant. Fc density on particles played a large role in complement system activation, both through the classical and alternative pathway, as it lead to a binary response for smaller particles and a tunable response for larger particles. We then applied these results to create a novel form of antibiotic by using Fc particles to direct complement-mediated bacterial cytotoxicity. The use of immune activation by Fc particles was also applied to better understand and improve the tuberculosis vaccine. Our findings are significant to the biomaterials and immunology fields as we showed that Fc microparticles can generally be used to alter the immune response in a tunable manner for a broad range of applications, as well answering fundamental immunology questions.

<p>The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.</p> / Dissertation